Synthesis and reactivity of potential toxic metabolites of tamoxifen analogues: Droloxifene and toremifene o-quinones

Article abstract of DOI:10.1021/tx010137i

Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormonedependent breast cancer. However, tamoxifen has been shown to increase the risk of endometrial cancer which has stimulated research for new effective antiestrogens, such as droloxifene and toremifene. In this study, the potential for these compounds to cause cytotoxic effects was investigated. One potential cytotoxic mechanism could involve metabolism of droloxifene and toremifene to catechols, followed by oxidation to reactive o-quinones. Another cytotoxic pathway could involve the oxidation of 4-hydroxytoremifene to an electrophilic quinone methide. Comparison of the amounts of GSH conjugates formed from 4-hydroxytamoxifen, droloxifene, and 4-hydroxytoremifene suggested that 4-hydroxytoremifene is more effective at formation of a quinone methide. However, all three substrates formed similar amounts of o-quinones. Both the tamoxifen-o-quinone and toremifene-o-quinone reacted with deoxynucleosides to give corresponding adducts. However, the toremifene-o-quinone was shown to be considerably more reactive than the tamoxifen-o-quinone in terms of both kinetic data as well as the yield and type of deoxynucleoside adducts formed. Since thymidine formed the most abundant adducts with the toremifene-o-quinone, sufficient material was obtained for characterization by ¹H NMR, COSY-NMR, DEPT-NMR, and tandem mass spectrometry. Cytotoxicity studies with tamoxifen, droloxifene, 4-hydroxytamoxifen, 4-hydroxytoremifene, and their catechol metabolites were carried out in the human breast cancer cell lines S30 and MDA-MB-231. All of the metabolites tested showed cytotoxic effects that were similar to the parent antiestrogens which suggests that o-quinone formation from tamoxifen, droloxifene, and 4-hydroxytoremifene is unlikely to contribute to their cytotoxicity. However, the fact that the o-quinones formed adducts with deoxynucleosides in vitro implies that the o-quinone pathway might contribute to the genotoxicity of the antiestrogens in vivo.

Full text of DOI:10.1021/tx010137i

Chem. Res. Toxicol. 2001, 14, 1643-1653
1643
Syn th esis a n d Rea ctivity of P oten tia l Toxic Meta bolites
of Ta m oxifen An a logu es: Dr oloxifen e a n d Tor em ifen e
o-Qu in on es
Dan Yao, Fagen Zhang, Linning Yu, Yanan Yang, Richard B. van Breemen, and
J udy L. Bolton*
Department of Medicinal Chemistry and Pharmacognosy (M/ C 781), College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231
Received August 15, 2001
Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormone-
dependent breast cancer. However, tamoxifen has been shown to increase the risk of
endometrial cancer which has stimulated research for new effective antiestrogens, such as
droloxifene and toremifene. In this study, the potential for these compounds to cause cytotoxic
effects was investigated. One potential cytotoxic mechanism could involve metabolism of
droloxifene and toremifene to catechols, followed by oxidation to reactive o-quinones. Another
cytotoxic pathway could involve the oxidation of 4-hydroxytoremifene to an electrophilic quinone
methide. Comparison of the amounts of GSH conjugates formed from 4-hydroxytamoxifen,
droloxifene, and 4-hydroxytoremifene suggested that 4-hydroxytoremifene is more effective at
formation of a quinone methide. However, all three substrates formed similar amounts of
o-quinones. Both the tamoxifen-o-quinone and toremifene-o-quinone reacted with deoxynucleo-
sides to give corresponding adducts. However, the toremifene-o-quinone was shown to be
considerably more reactive than the tamoxifen-o-quinone in terms of both kinetic data as well
as the yield and type of deoxynucleoside adducts formed. Since thymidine formed the most
abundant adducts with the toremifene-o-quinone, sufficient material was obtained for
characterization by 1H NMR, COSY-NMR, DEPT-NMR, and tandem mass spectrometry.
Cytotoxicity studies with tamoxifen, droloxifene, 4-hydroxytamoxifen, 4-hydroxytoremifene,
and their catechol metabolites were carried out in the human breast cancer cell lines S30 and
MDA-MB-231. All of the metabolites tested showed cytotoxic effects that were similar to the
parent antiestrogens which suggests that o-quinone formation from tamoxifen, droloxifene,
and 4-hydroxytoremifene is unlikely to contribute to their cytotoxicity. However, the fact that
the o-quinones formed adducts with deoxynucleosides in vitro implies that the o-quinone
pathway might contribute to the genotoxicity of the antiestrogens in vivo.
DNA (8). Concerns about the side effects of tamoxifen
have stimulated a search for new effective agents that
do not form such genotoxic species.
In tr od u ction
Tamoxifen1 is currently the standard treatment for
hormone dependent breast cancer in postmenopausal
women (1-4). However, tamoxifen has been shown to
cause an increased incidence of hepatocellular tumors in
rats (5, 6) and an increase in the risk of endometrial
cancer in women (7). A potential genotoxic mechanism
might involve metabolic activation by oxidative enzymes
in vivo to an electrophile(s) that binds irreversibly to
Toremifene has been recently approved by the United
States Food and Drug Administration for the treatment
of advanced breast cancer in postmenopausal women (9).
There have been several recent reports comparing the
efficacy and metabolism of tamoxifen and toremifene
(10-19). Although toremifene is structurally similar to
tamoxifen, differing only by a single chlorine atom in the
ethyl side chain, toremifene does not cause hepatocar-
cinogenesis in rats in vivo and it produces considerably
fewer DNA adducts (20). Droloxifene (3-hydroxytamox-
ifen) is another antiestrogen, which has shown promise
in the treatment of breast cancer (21-23). There is no
evidence that droloxifene produces DNA adducts or
hepatocelluar carcinoma in rats (7, 24).
There are several metabolic activation pathways of
tamoxifen and toremifene leading to DNA adducts such
as formation of carbocations (20, 25) and electrophilic
quinone methides (26-28). In addition, an o-quinone has
been shown to be a potential reactive intermediate
involved in the metabolism of both droloxifene and
toremifene (29, 30). In the present study, we compared
* To whom correspondence should be addressed. Phone: (312) 996-
5280. Fax: (312) 996-7107. E-mail: judy.bolton@uic.edu.
1 Abbreviations: tamoxifen, Z-2-[4-(1,2-diphenyl-1-butenyl)-phenoxy]-
N,N-dimethylethanamine; toremifene, Z-2-[(4-chloro-1,2-diphenyl-1-
butenyl)-phenoxy]-N,N-dimethylethanamine; 4-OHTAM, 4-hydroxyta-
moxifen; 3,4-di-OHTAM, 3,4-dihydroxytamoxifen; 4-OHTAM-SG,
glutathione conjugates of 4-hydroxytamoxifen; 3,4-di-OHTAM-diSG,
di-glutathione conjugates of 3,4-dihydroxytamoxifen; 4-OHTOR, 4-hy-
droxytoremifene; 3,4-di-OHTOR, 3,4-dihydroxytoremifene; 4-OHTOR-
diSG, di-glutathione conjugates of 4-hydroxytoremifene; 3,4-di-OHTOR-
diSG, di-glutathione conjugates of 3,4-dihydroxytoremifene; 3,4-di-
OHTOR-triSG, tri-glutathione conjugates of 3,4-dihydroxytoremifene;
3,4-di-OHTOR-T, thymidine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR-
dG, deoxyguanosine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR-dA,
deoxyadenosine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR-dC, deoxy-
cytosine adducts of 3,4-di-OHTOR; P450, cytochrome P450; GSH,
glutathione; electrospray-MS, electrospray mass spectrometry; MS-
MS, tandem mass spectrometry; ER, estrogen receptor.
10.1021/tx010137i CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/27/2001

1644 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Yao et al.
methanol over the next 39 min, and finally increased to 95%
methanol over the remaining 5 min. Method D: LC-MS-MS
analysis was performed using a 4.6 × 150 mm Ultrasphere C-18
column with on-line UV absorbance detection at 280 nm and
electrospray MS-MS detection. The mobile phase consisted of
25% methanol in 0.5% ammonium acetate (pH 3.5) at a flow
rate of 1.0 mL/min for 5 min, which was increased to 35%
methanol over 1 min, then to 50% methanol over the next 39
min, and finally to 95% methanol over the remaining 10 min.
Method E: LC-MS-MS analysis was the same as described in
Method D with a modified mobile phase. The mobile phase
consisted of 30% methanol in 0.5% ammonium acetate (pH 3.5)
at a flow rate of 1.0 mL/min for 10 min, which was increased to
48% methanol over the next 35 min, then to 95% methanol over
the remaining 20 min. Method F: A semipreparative isocratic
method was developed using a 10 × 250 mm Ultrasphere C18
column with a mobile phase consisting of 46% methanol in 0.5%
acetic acid (pH 3.5) at a flow rate of 3.0 mL/min and detection
at 280 nm.
the potential toxicity of quinoid intermediates formed
from tamoxifen analogues, including those of 4-hydroxy-
tamoxifen, droloxifene, and 4-hydroxytoremifene. Two
human breast cancer cell lines, S30 (ER-positive) and
MDA-MB-231 (ER-negative), were used to study the
relative cytotoxicity of 4-hydroxytamoxifen, droloxifene,
3,4-dihydroxytamoxifen, 4-hydroxytoremifene, and 3,4-
dihydroxytoremifene. The data suggest that o-quinone
formation might not contribute to the cytotoxic mecha-
nism of the antiestrogens. However, the formation of
DNA adducts implies that the o-quinone pathway might
contribute to the genotoxic effects of these antiestrogens
in vivo.
Ma ter ia ls a n d Meth od s
Ca u tion : Toremifene-o-quinone was handled in accordance
with the NIH Guidelines for the Laboratory Use of Chemical
Carcinogens (31). All solvents and chemicals were purchased
from either Aldrich Chemical Co. (Milwaukee, WI) or Fisher
Scientific (Itasca, IL) unless stated otherwise. [3H]GSH (glycine-
2-3H) was obtained from New Life Science Products Inc. (Boston,
MA) and diluted to a specific activity of 40 nCi/nmol. 4-Hydroxy-
tamoxifen and 4-hydroxytoremifene were synthesized as de-
scribed previously (32). Droloxifene was synthesized according
to the literature procedure (33).
In str u m en ta tion . 1H NMR and 13C NMR spectra were
obtained with a Bruker (Billerica, MA) Avance DPX300 spec-
trometer at 300 MHz. UV spectra were measured on a Hewlett-
Packard (Palo Alto, CA) 8452A photodiode array UV-vis
spectrophotometer. Exact mass measurements were obtained
using EI or CI on a Fininigan (Bremen, Germany) MAT90
magnetic sector mass spectrometer at a resolving power of
10 000. LC-MS was carried out with a Hewlett-Packard 5989B
mass spectrometer equipped with an Analytica (Branford, CT)
electrospray ion source and ion guide. LC-MS-MS spectra were
obtained using a Micromass (Manchester, U.K.) Quattro II triple
quadrupole mass spectrometer equipped with a Waters (Milford,
MA) 2487 UV detector and a Waters 2690 HPLC system.
Collision-induced dissociation (CID) was carried out using a
range of collision energies from 25 to 70 eV and an argon
collision gas pressure of 2.7 µbar. HPLC experiments were
performed on a Shimadzu (Columbia, MD) LC-10A gradient
HPLC system equipped with a SIL-10A auto injector, an SPD-
M10AV UV-vis photodiode array detector, and an SPD-10AV
detector.
Syn th esis of 3,4-Dih yd r oxytor em ifen e (Sch em e 1). 4-[2-
(N,N-Dimethylamino)ethoxy]-3,4-methylenedioxybenzophenone
1. 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone was syn-
thesized as described previously (34). To a 250 mL flask were
combined 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone
(3.55 g, 11.6 mmol) and dimethylamine (2.0 M solution in
methanol, 60 mL, 0.12 mol), and the mixture was refluxed under
N2 for 5 days. The reaction mixture was concentrated and the
residue was partitioned between diethyl ether (80 mL) and 0.1
M aqueous NaOH (50 mL). The aqueous layer was extracted
with ether, and the combined organic layers were washed with
water and dried over anhydrous Na2SO4. After concentration,
the residue was purified by column chromatography (silica gel)
using ethyl acetate/triethylamine (3:1, v/v) as eluent to give
compound 1 (2.60 g, 70% yield). 1H NMR (CDCl3) δ 2.37 (s, 6H,
CH3), 2.78 (t, 2H, J ) 5.7 Hz, NCH2), 4.16 (t, 2H, J ) 5.7 Hz,
OCH2), 6.07 (s, 2H, OCH2O), 6.87 (d, 1H, J ) 8.9 Hz, ArH), 6.99
(d, 2H, J ) 8.9 Hz, ArH), 7.32 (d, 2H, J ) 8.9 Hz, ArH), 7.77(d,
2H, J ) 8.9 Hz, ArH); 13C NMR (CHCl3) δ 46.3, 58.5, 66.6, 102.1,
108.0, 110.3, 114.4, 126.6, 131.0, 132.6, 132.9, 148.2, 151.5,
162.6, 194.4. (E,Z)-1-{4-[2-(N,N-Dimethylethylamino)ethoxy]phe-
nyl}-1-(3,4-methylenedihdroxyphenyl)-2-phenyl-4-chlorobut-1-
ene 2. TiCl4 (2.85 g, 15.0 mmol) was added dropwise to a stirred
suspension of zinc powder (1.96 g, 30.0 mmol) in THF (35 mL)
at -10 °C under N2. The resulting mixture was heated at reflux
for 1.5 h. The suspension was cooled to room temperature and
a mixture of compound 1 (1.10 g, 3.5 mmol) and 3-chloropro-
piophone (0.60 g, 3.5 mmol) in dry THF (30 mL) was added
dropwise. The mixture was refluxed for 4 h, cooled to room
temperature, and combined with 10% aqueous potassium
carbonate (80 mL). The resulting mixture was stirred for 5 min
and extracted with ethyl acetate (3 × 150 mL). The combined
organic extracts were washed with saturated NaCl, dried over
anhydrous Na2SO4, and concentrated. The final residue was
purified by column chromatography (silica gel) using ethyl
acetate/triethylamine (20:1, v/v) as eluent to give compound 2
as a mixture of E (trans) and Z (cis) isomers (2:5 E:Z) (0.85 g,
54% yield): 1H NMR (CD3OD) δ 2.29 (s, 6H, trans N(CH3)2),
2.35 (s, 6H, cis N(CH3)2), 2.68 (t, 2H, J ) 5.4 Hz, trans CH2N),
2.79 (t, 2H, J ) 5.4 Hz, cis CH2N), 2.94 (m, 4H, cis and trans
CH2), 3.42 (m, 4H, cis and trans CH2Cl), 3.96 (t, 2H, J ) 5.4
Hz, trans CH2O), 4.11 (t, 2H, J ) 5.4 Hz, cis CH2O), 5.77 (s,
1H, trans OCHO), 5.95 (s, 1H, cis OCHO), 6.34 (d, 1H, J ) 1.3
Hz, ArH), 6.37 (d, 1H, J ) 8.8 Hz, ArH), 6.48 (d, 1H, J ) 8.8
Hz, ArH), 6.82 (d, 2H, J ) 8.8 Hz, ArH), 6.97 (d, 2H, J ) 8.8
Hz, ArH), 7.15-7.23 (m, 5H, ArH); EI-MS (m/z) 449 (100%)
[M]+., 451 (38%) [M + 2]+. (E,Z)-1-{4-[2-(N,N-Dimethylethylami-
no)ethoxy]phenyl}-1-(3,4-dihyoxyphenyl)-2-phenyl-4-chlorobut-1-
ene 3 (34). To a flame-dried flask were added compound 2 (0.84
g, 1.87 mmol), 1 M solution of boron trichloride in methylene
chloride (15.0 mL, 15.0 mmol), and 1,2-dichloroethane (35 mL)
under N2. The solution was stirred for 48 h at room temperature.
MeOH (45 mL) was gradually added to the solution, and the
HP LC Meth od ology. Six general methods were used to
analyze and separate metabolites, GSH conjugates, and deoxy-
nucleoside adducts. All retention times mentioned in the text
were obtained using Method A unless stated otherwise. Method
A: Analytical HPLC analysis was performed using a 4.6 × 150
mm Ultrasphere C18 column (Beckman, San Ramon, CA) using
a Shimadzu HPLC system with UV detection at 280 nm. The
mobile phase consisted of 20% methanol in 0.25% acetic acid
and 0.25% perchloric acid (pH 3.5) at a flow rate of 1.0 mL/min
for 5 min, increased to 35% methanol over 1 min, then to 60%
methanol over the course of the next 39 min, and finally
increased to 95% methanol over the remaining 5 min. Method
B: A semipreparative method was developed using a 10 × 250
mm Ultrasphere C18 column and a mobile phase consisting of
40% methanol in 0.25% acetic acid and 0.25% perchloric acid
(pH 3.5) at a flow rate of 3.0 mL/min for 5 min, increased to
50% methanol over the next 6 min, isocratic for the following
29 min, increased to 54% methanol over the next 15 min, and
finally to 95% methanol over the last 5 min of the run. Method
C: LC-MS analysis was performed using a 4.6 × 150 mm
Ultrasphere C18 column with
a photodiode array UV-vis
absorbance detector set at 230-350 nm and a electrospray mass
spectrometer. The mobile phase consisted of 20% methanol in
0.5% ammonium acetate (pH 3.5) at a flow rate of 1.0 mL/min
for 5 min, increased to 35% methanol over 1 min, then to 60%

Droloxifene- and Toremifene-o-quinones
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1645
Sch em e 1. Syn th esis of 3,4-Dih yd r oxytor em ifen ea
a
Reagents and conditions: (a) dimethylamine, reflux, 5 days; (b) TiCl4, Zn, 3-chloropropiophenone, THF, reflux, 4 h; (c) BCl3, 1,2-
dichloroethane, rt, 48 h.
mixture was stirred for 40 min. The solvent was removed under
reduced pressure and a saturated NaHCO3 solution (50 mL) and
MeOH (7 mL) was added to the residue. The pH of the solution
was adjusted to 9.0 with 1 N NaOH and the resulting solution
was extracted with ethyl acetate (2 × 150 mL). The combined
organic layers were washed with saturated NaHCO3 (50 mL),
H2O (100 mL), and dried over anhydrous sodium sulfate. After
filtration, the solvent was removed and the final residue was
purified by flash chromatography (silica gel) using methanol/
2-propanol/acetic acid (12:2:1, v/v/v) as eluent to give compound
3 as a mixture of E (trans) and Z (cis) isomers (5:8 E:Z) (0.35 g,
43% yield): 1H NMR (CD3OD) δ 2.71 (s, 6H, trans N(CH3)2),
2.78 (s, 6H, cis N(CH3)2), 2.90 (t, 2H, J ) 7.3 Hz, trans CH2Cl),
2.96 (t, 2H, J ) 7.3 Hz, cis CH2Cl), 3.40 (t, 4H, cis and trans
CH2N), 4.12 (t, 2H, J ) 5.1 Hz, trans CH2O), 4.30 (t, 2H, J )
5.1 Hz, cis CH2O), 6.18 (d, 1H, J ) 8.1 Hz, ArH), 6.32 (d, 1H, J
) 2.0 Hz, ArH), 6.38 (d, 1H, J ) 8.1 Hz, ArH), 6.64 (d, 2H, J )
8.6 Hz, ArH), 6.77 (d, 1H, J ) 8.0 Hz, ArH), 6.83 (d, 1H, J )
8.7 Hz, ArH), 7.02 (d, 2H, J ) 8.7 Hz, ArH), 7.14-7.20 (m, 13H,
ArH), 7.24 (d, 2H, J ) 8.7 Hz, ArH); CI-MS (positive ion,
methane) m/z 438 (100%) [M + H]+.
P r ep a r a tion of Tor em ifen e-3,4-qu in on e. Activated silver
oxide (35) was prepared by adding KOH (0.72 g in 20 mL of
H2O) to AgNO3 solution (2.0 g in 20 mL of H2O). The mixture
was stirred for 15 min, the precipitate was washed with H2O,
filtered, and dried. A mixture of 3,4-dihydroxytoremifene (4.4
mg), fresh silver oxide (440 mg), and acetonitrile (4 mL) was
stirred for 15 min at 60 °C. After filtration, the solution was
concentrated to a final volume of 1 mL. An aliquot (25 µL) of
this solution was analyzed by LC-MS (Method A). Toremifene-
o-quinone: UV (CH3OH/H2O) 227, 265, 375, 460 nm; positive
ion electrospray MS m/z 436 (100%) [M + H]+; retention time
40 min.
Method B. 3,4-di-OHTOR-SG 1: UV (CH3/H2O) 245, 270, 340
nm; 1H NMR (CD3OD) δ 2.10 (m, 2H, Gluâ), 2.44 (m, 2H, Gluγ),
2.80 (m, 1H, cysâ), 2.92 (t, 2H, J ) 7.6 Hz, CH2), 3.03 (s, 6H,
CH3N), 3.07 (m, 1H, cysâ), 3.42 (t, 2H, J ) 7.6 Hz, CH2Cl), 3.60
(t, 2H, J ) 4.9 Hz, CH2N), 3.64 (m, 1H, GluR), 3.78 (d, 2H, GlyR),
4.23 (m, 1H, cysR), 4.40 (t, 2H, J ) 4.9 Hz, OCH2), 6.34 (d, 1H,
J ) 2.0 Hz, ArH), 6.41 (d, 1H, J ) 2.0 Hz, ArH), 7.06 (d, 2H, J
) 8.7 Hz, ArH), 7.13-7.27 (m, 5H, ArH), 7.29 (d, 2H, J ) 8.7
Hz, ArH); positive ion electrospray MS m/z 743 (100%) [M +
H]+; MS-MS with CID of m/z 725 (30%) [MH - H2O]+, 614
(100%) [MH - Glu]+, 511 (5%) [3,4-dihydroxytoremifene +
SCH2CHdNH2]+, 470 (60%) [3,4-dihydroxytoremifene + SH]+;
retention time 34 min. 3,4-di-OHTOR-SG 2: UV (CH3OH/H2O)
248, 277, 325, 343 nm; 1H NMR (CD3OD) δ 2.10 (m, 2H, Gluâ),
2.44 (m, 2H, Gluγ), 2.92 (s, 6H, CH3N), 2.99 (t, 2H, J ) 7.2 Hz,
CH2), 3.04 (m, 1H, cysâ), 3.41 (t, 2H, J ) 7.2 Hz, CH2Cl), 3.50
(t, 2H, J ) 4.7 Hz, CH2N), 3.62 (m, 1H, GluR), 3.72 (d, 2H, GlyR),
4.23 (t, 2H, J ) 4.7 Hz, CH2O), 4.35 (m, 1H, cysR), 6.69 (d, 2H,
J ) 8.8 Hz, ArH), 6.75 (d, 1H, J ) 2 Hz, ArH), 6.81 (d, 1H, J )
2 Hz, ArH), 6.87 (d, 2H, J ) 8.8 Hz, ArH), 7.04-7.19 (m, 5H,
ArH); positive ion electrospray MS m/z 743 (100%) [M + H]+;
MS-MS with CID of m/z 725 (25%) [MH - H2O]+, 614 (97%)
[MH - Glu]+, 511 (8%) [3,4-dihydroxytoremifene + SCH2CHd
NH2]+, 470 (100%) [3,4-dihydroxytoremifene + SH]+; retention
time 36 min. 3,4-di-OHTOR-SG 3: UV (CH3OH/H2O) 243, 263,
322, 342 nm; positive ion electrospray MS m/z 743 (100%); MS-
MS with CID of m/z 725 (8%) [MH - H2O]+, 614 (75%) [MH -
Glu]+, 511 (3%) [3,4-dihydroxtoremifene + SCH2CHdNH2]+, 470
(45%) [3,4-dihydroxytoremifene + SH]+; retention time 40 min.
3,4-di-OHTOR-diSG: UV (CH3OH/H2O) 234, 273, 314 nm;
positive ion electrospray MS m/z 525 (100%) [M + 2H]2+
;
retention time 30 min.
Oxid a tion of 3,4-Dih yd r oxytor em ifen e by Tyr osin a se.
A mixture of 3,4-dihydroxytoremifene (0.5 mM), tyrosinase (0.5
mg/mL), and GSH (5 mM) in 50 mM phosphate buffer (pH 7.4,
2.0 mL of total volume) was incubated for 30 min at 37 °C.
Reactions were terminated by chilling in an ice bath followed
by the addition of perchloric acid (50 µL/mL). Control incuba-
tions were conducted without tyrosinase or GSH. The reaction
mixtures were centrifuged at 13 000 rpm for 6 min and the
solutions were extracted using solid-phase extraction cartridges
Rea ction of Tor em ifen e-o-qu in on e w ith GSH. A solution
of toremifene-o-quinone (0.01 M) in acetonitrile was combined
with GSH (0.1 M) in incubation buffer (6 mL, 50 mM phosphate
buffer, pH 7.4), and the mixture was stirred at room tempera-
ture for 5 min. Perchloric acid (1.2 mL) was added, and the
solution was concentrated to remove the remaining acetonitrile
under a stream of N2. The final solution was centrifuged for 6
min at 13 000 rpm. The conjugates were purified by using

1646 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Yao et al.
(Waters Oasis, HLB), eluted with methanol, and concentrated
to a final volume of 250 µL. Aliquots (50 µL) were analyzed
directly by LC-MS using Method C. In addition to the mono-
and di-GSH conjugates described above, tri-GSH conjugates
were also obtained. 3,4-di-OHTOR-triSG 1: UV (CH3OH/H2O)
225, 270, 325 nm; positive ion electrospray MS m/z 678 (100%)
[M + 2H]2+; retention time 14 min. 3,4-di-OHTOR-triSG 2: UV
(CH3OH/H2O) 232, 272, 320 nm; positive ion electrospray MS
m/z 678 [M + 2H]2+; retention time 15 min.
P r ep a r a tion of Ra t Liver Micr osom es. Female Sprague-
Dawley rats (200-220 g) were obtained from Sasco Inc. (Omaha,
NE). The rats were pretreated with dexamethasone to induce
P450 3A isozymes. They were given intraperitoneal injections
of dexamethasone in corn oil (100 mg/kg) daily for 3 consecutive
days and were sacrificed on day 4. Rat liver microsomes were
prepared and protein and P450 concentrations were determined
as described previously (36).
F igu r e 1. Time course analysis of incubation of tamoxifen-o-
quinone (squares) and toremifene-o-quinone (circles) at 37 °C
and pH 7.4. Aliquots (200 µL) were combined with 5.0 mM GSH
and 10 µL of PCA at various times. The data represent the peak
area ratios of the GSH conjugates relative to that of the internal
standard (2,4-dihydroxybenzoic acid).
Oxidation of 4-Hydr oxytam oxifen , 4-Hydr oxytor em ifen e,
a n d Dr oloxifen e by Ra t Liver Micr osom es. A solution
containing the substrate (0.1 mM), rat liver microsomes (1 nmol
P450), [3H]GSH (specific activity of 40 nCi/nmol, 2.5 mM), and
a NADPH-generating system (including 1.0 mM NADP+, 5.0
mM MgCl2, 5.0 mM isocitric acid, and 0.2 unit/mL isocitric
dehydrogenase) in 50 mM phosphate buffer (pH 7.4, 0.5 mL total
volume) was incubated for 30 min at 37 °C (37). For control
incubations, NADP+ was omitted. The reactions were termi-
nated by chilling in an ice bath followed by the addition of
perchloric acid (25 µL). Radioactivity eluting from the HPLC
column was measured in fractions collected at 18 s intervals.
(Northwestern University, Evanston, IL) and was maintained
in MEME medium supplemented with 5% charcoal-dextran-
treated fetal bovine serum, 1% gluta-max, 1% penicillin, strep-
tomycin, fungizome, 1% nonessential amino acids, 500 µg/mL
G418, 6 mg/L insulin, and 5% CO2 (39). MDA-MB-231 cells were
maintained in Liebovitz L-15 medium supplemented with 1%
penicillin, streptomycin, fungizome, 1% gluta-max, 6 mg/L
insulin, 5% fetal bovine serum, and 5% CO2 (39). The medium
was routinely changed every 3 or 4 days and was changed 24 h
prior to each experiment to maintain logarithmic growth. The
cells were treated for 18 h with each test compound at final
concentrations ranging from 5 to 100 µM. Negative controls
(cells treated with DMSO only) were also carried out to define
100% cell viability. After treatment, floating cells were collected,
attached cells were first trypsinized, and then floating and
attached cells were combined. All cells were harvested by
centrifugation at 2000 rpm for 5 min. Trypan blue stain (0.1
mL of 0.4%) was added to each cell suspension at a total volume
of 0.6 mL. The cell suspension was transferred to a hemacy-
tometer and the number of cells was counted under a micro-
scope. The dead cells were stained blue and viable cells excluded
the stain. The LC50 values were obtained by regression and
linear estimation analysis. The data represent the means ( SD
of three determinations.
Rea ction of Tor em ifen e-o-qu in on e w ith Deoxyn u cleo-
sid es. A solution of toremifene-o-quinone (0.5 mM) in acetoni-
trile was combined with four deoxynucleosides (25 mM each)
in phosphate buffer (pH 7.4) in a total volume of 5.0 mL. The
solution was incubated for 5 h at 37 °C, extracted using Oasis
solid-phase extraction cartridges, eluted with methanol, and
concentrated to a final volume of 250 µL. Aliquots (50 µL) were
analyzed directly by LC-MS-MS (Methods D and E). Toremifene-
o-quinone was shown to react with all four deoxynucleosides.
Three adducts were obtained with thymidine, five adducts were
detected with deoxyguanosine, two adducts were observed with
deoxyadenosine, and eight adducts were obtained with deoxy-
cytosine. The 3,4-dihydroxytoremifene thymidine adducts all
gave similar positive ion electrospray mass spectra with proto-
nated molecules at m/z 678. The retention times were 24.0, 28.5,
and 29.8 min (Method D). 3,4-di-OHTOR-T 1 was purified by
semipreparative HPLC (Method F). 3,4-di-OHTOR-T 1: 1H
NMR (DMSO-d6) δ 1.91 (s, 3H, CH3-T), 2.23 (s, 6H, N(CH3)2),
2.63 (t, 2H, J ) 5.7 Hz, NCH2), 2.85 (t, 2H, CH2), 3.74 (m, 2H,
5′-H-T), 3.83 (m, 1H, 4′-H-T), 4.08 (m, 1H, 3′-H-T), 4.25 (t, 1H,
5′-OH-T, D2O exchangeable), 5.63 (t, 1H, J ) 6.9 Hz, 1′-H-T),
6.27 (s, 1H, 2-OH, D2O exchangeable), 6.28 (s, 1H, 1-OH, D2O
exchangeable), 6.36 (d, 1H, J ) 2.1 Hz, ArH), 6.39 (d, 1H, J )
1.8 Hz, ArH), 6.69 (d, 2H, J ) 8.0 Hz, ArH), 6.86 (d, 2H, J )
8.0 Hz, ArH), 7.16-7.20 (m, 5H, ArH), 7.58 (s, 1H, 6-H-T); MS-
MS with CID of m/z 678 (22%) [M + H]+, 562 (100%) [MH -
deoxyribose]+, 72 (21%) [CH2CH2N(CH3)2]+; retention time 24.0
min (Method D). The 3,4-dihydroxytoremifene deoxyguanosine
adducts all gave similar positive ion electrospray mass spectra
with protonated molecules at m/z 703. The retention times were
23.8, 29.0, 30.1, 32.0, and 41.5 min (Method E). 3,4-di-OHTOR-
dG 1: MS-MS with CID of m/z 703 (15%) [M + H]+, 587 (100%)
[MH - deoxyribose]+. The 3,4-dihydroxytoremifene deoxyad-
enosine adducts all gave similar positive ion electrospray mass
spectra with protonated molecules at m/z 687. The retention
times were 23.8 and 25.6 min (Method E). The 3,4-dihydroxy-
toremifene deoxycytosine adducts also gave similar positive ion
electrospray mass spectra with protonated molecules at m/z 663.
The retention times were 18.9, 23.1, 25.8, 31.6, 34.2, 35.3, 37.0,
and 38.7 min (Method E).
Resu lts a n d Discu ssion
Rea ctivity of Tor em ifen e-o-qu in on e. Among the
chemical and enzymatic agents that have been reported
to oxidize catechols including manganese dioxide, lead
(IV) oxide, and tyrosinase (40, 41), we used silver oxide
(35) as the chemical oxidizing agent and tyrosinase as
the enzymatic agent to generate the o-quinone of 3,4-
dihydroxytoremifene. The UV spectra showed typical
o-quinone chromophores at 375 and 460 nm. The lifetime
of the toremifene-o-quinone under physiological condi-
tions was determined by removing aliquots at various
times and “quenching” it with GSH. The reaction system
at each time was analyzed by HPLC to give a series of
chromatograms with time-dependent decreased peak
area of the o-quinone GSH conjugates (Figure 1). Under
these conditions, the half-life of the toremifene-o-quinone
was determined to be approximately 8 min. Similar
studies with tamoxifen-o-quinone gave a half-life of about
80 min. The much shorter half-life of toremifene-o-
quinone compared to tamoxifen-o-quinone suggests that
the toremifene-o-quinone is less stable and more reactive
than tamoxifen-o-quinone. The reason for the increased
reactivity could be due to the chlorine substituent which
makes the toremifene-o-quinone more electrophilic com-
pared to tamoxifen-o-quinone.
Cytotoxicity Stu d ies in S30 a n d MDA-MB-231 Cells. The
trypan blue exclusion assay was conducted to determine cell
viability (38). The S30 cell line was provided by Dr. V. C. J ordan

Droloxifene- and Toremifene-o-quinones
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1647
F igu r e 2. Positive ion electrospray CID MS-MS of m/z 743 of 3,4-di-OHTOR-SG 2.
pathway for these antiestrogens (42, 43). In the current
3,4-Dihydroxytoremifene-o-quinone formed using silver
oxide reacted with GSH to give four GSH conjugates, one
di-GSH conjugate, and three mono-GSH conjugates
(Scheme 2). When tyrosinase was used as the oxidizing
agent, two additional tri-GSH conjugates were obtained
likely due to further oxidation of the mono and di
conjugates in the presence of the enzyme. During MS-
MS with CID of the protonated molecules of m/z 743,
all of the mono GSH conjugates showed similar fragmen-
tation patterns. For example, all formed dehydrated ions
of m/z 725 (Figure 2). The ion at m/z 614 represented
the loss of 129 mass units (pyroglutamic acid), and the
ions of m/z 511, 470, and 72 corresponded to [3,4-
dihydroxytoremifene + SCH2CHdNH2]+, [3,4-dihydroxy-
toremifene + SH]+, and [dimethylaziridinium, CH2CH2N-
(CH3)2]+, respectively. The di-GSH conjugate 3,4-di-
OHTOR-di-SG gave a doubly charged ion at m/z 525 [M
+ 2H]2+. The mono-GSH conjugates were sufficiently
stable for HPLC purification and their structures were
elucidated with 1H NMR and COSY-NMR. We predict
that the GSH molecule reacts at the C6 position on the
catechol ring to give the E/Z isomers 3,4-di-OHTOR-SG
1 and 3,4-di-OHTOR-SG 2. In support of this, we
study, it was of interest to determine the relative ability
of cytochrome P450 to convert droloxifene, 4-hydroxyta-
moxifen, and 4-hydroxytoremifene to o-quinones. We used
microsomes isolated from female Sprague-Dawley rats
treated with dexamethasone to induce P450 3A isozymes.
The results showed that both droloxifene and 4-hydroxy-
tamoxifen were oxidized to tamoxifen-o-quinone which
reacted with GSH to give GSH conjugates. Previously it
has been shown that tamoxifen-o-quinone binds co-
valently to proteins and DNA (44). In addition to the
o-quinone, we and others (45) have shown that 4-hydroxy-
tamoxifen is also oxidized to 4-hydroxytamoxifen quinone
methide (Scheme 3). Previous work (46) showed that
4-hydroxytamoxifen quinone methide could react with
GSH either through 1,8-Michael addition or 1,6-Michael
addition to give GSH adducts. In contrast, droloxifene,
which has a hydroxy group at the C3 position of the
phenyl ring, cannot be converted to a quinone methide.
As a result, only 3,4-dihydroxytamoxifen GSH conjugates
and unreacted 3,4-dihydroxytamoxifen were detected in
microsomal incubations with droloxifene (Scheme 3).
Incubations with 4-hydroxytoremifene gave similar re-
sults as 4-hydroxytamoxifen in that two species of quinoid
metabolites were formed: 4-hydroxytoremifene quinone
methide and toremifene-o-quinone (Scheme 4). We have
previously shown that 4-hydroxytoremifene quinone me-
thide reacted with two molecules of GSH to produce the
corresponding two isomers of di-GSH conjugates as
shown in Scheme 4 (46). The reaction mechanism prob-
ably involves formation of an episulfonium ion intermedi-
ate which might contribute to the potential cytotoxic
effects of toremifene. In the present study, three ad-
ditional peaks were observed in the HPLC analysis of
4-hydroxytoremifene incubations, which were identified
as the 3,4-dihydroxytormifene di-GSH conjugate and 3,4-
dihydroxytoremifene mono-GSH conjugates. Their reten-
tion times were identical to those obtained when 3,4-
dihydroxytoremifene was used as the substrate. 3,4-
Dihydroxytoremifene was also observed in the HPLC
chromatogram of the 4-hydroxytoremifene incubation
1
observed two doublets in the aromatic region of the H
NMR spectra which represent the meta coupling (J )
2.0 Hz) between the H-3 proton and H-5 protons, respec-
tively. Unfortunately, the di-GSH conjugates were not
sufficiently stable for the HPLC purification and NMR
characterization. The reason for the instability of the di-
GSH conjugates is likely due to the additional electron-
donating sulfur substituent which facilitates oxidation
of the aromatic ring, producing initially the o-quinone
di-GSH conjugate which can further react with other
nucleophiles in solution or undergo dimerization reac-
tions. The tri-GSH conjugates obtained with tyrosinase
were also not stable and only their MS and UV data were
obtained.
Oxid a tion of 4-Hyd r oxyta m oxifen , Dr oloxifen e,
an d 4-Hydr oxytor em ifen e by Rat Liver Micr osom es.
It is well established that 4-hydroxylation of both tamox-
ifen and toremifene represents a major phase I metabolic

1648 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Sch em e 2. Str u ctu r es of th e GSH Con ju ga tes of Tor em ifen e-o-qu in on e
Yao et al.
Sch em e 3. Meta bolism of 4-Hyd r oxyta m oxifen a n d Dr oloxifen e to Qu in oid s a n d Rea ction w ith GSH
system. We determined the amount of each quinoid
formed from 4-hydroxytamoxifen, droloxifene, and 4-hy-
droxytoremifene in rat liver microsomes by trapping the
electrophiles with [3H]GSH. The values shown in Tables
1-3 might underestimate quinoid metabolite formation,
because of the possibility that other nucleophiles in the
incubation system, such as microsomal proteins, might
bind to the quinoid metabolites. The radiochromatograms
gave peaks with retention times identical to those
obtained from GSH conjugation with the synthetic o-
quinones or quinone methides (Table 1-3). In the 4-hy-
droxytamoxifen incubation (Table 1), the amount of
quinone methide-derived GSH conjugates was 0.20 nmol
(nmol of P450)-1 (10 min)-1 and the amount of o-quinone-
derived GSH conjugates was 0.49 nmol (nmol of P450)-1
(10 min)-1. The increased amount of o-quinone relative
to quinone methide may suggest that aromatic hydroxy-
lation followed by oxidation to an o-quinone is preferred
for 4-hydroxytamoxifen compared to direct oxidation to
a quinone methide. In the 4-hydroxytoremifene incuba-
tion (Table 2), the amount of quinone methide-derived
GSH conjugates was higher than that of o-quinone-
derived GSH conjugates. The amount of quinone methide
formed from 4-hydroxytoremifene was 3-fold higher than
4-hydroxytamoxifen perhaps due to the â-Cl substitution
which increased the acidity of the R-H and eased 2-elec-
tron oxidation forming 4-hydroxytormifene quinone me-
thide. Previous work examining the effect of ring sub-

Droloxifene- and Toremifene-o-quinones
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1649
Sch em e 4. Meta bolism of 4-Hyd r oxytor em ifen e to Qu in oid s a n d Rea ction w ith GSH
Ta ble 1. Con ver sion of 4-Hyd r oxyta m oxifen to GSH Con ju ga tes by Ra t Liver Micr osom esa
retention
time (min)
rate of formation
quinoid
conjugate
[nmol (nmol of P450)-1 (10 min)-1
]
quinone methide
4-OHTAM-SG 1
4-OHTAM-SG 2 and 3
4-OHTAM-SG 4
25
28
30
0.052 ( 0.007
0.089 ( 0.006
0.058 ( 0.013
total: 0.20 ( 0.03
0.211 ( 0.028
o-quinone
3,4-di-OHTAM-diSG 2
3,4-di-OHTAM-SG 1
3,4-di-OHTAM-SG 2
3,4-di-OHTAM-SG 3
31
34
37
40
0.073 ( 0.014
0.111 ( 0.019
0.090 ( 0.015
total: 0.49 ( 0.08
a
Incubations were conducted for 30 min with 0.1 mM substrate and rat liver microsomes (1.0 nmol of P450/mL) in the presence of an
NADPH-generating system and 2.5 mM [3H]GSH (specific activity of 40 nCi/nmol) at 37 °C. Radioactivity eluting from the HPLC column
was measured in fractions collected at 18 s intervals. Results are the average ( SD of three incubations.
Ta ble 2. Con ver sion of 4-Hyd r oxytor em ifen e to GSH Con ju ga tes by Ra t Liver Micr osom esa
retention
time (min)
rate of formation
quinoid
conjugate
[nmol (nmol of P450) -1 (10 min) -1
]
quinone methide
4-OHTOR-diSG 1 and 2
4-OHTOR-diSG 3 and 4
13
16
0.24 ( 0.03
0.42 ( 0.11
total: 0.66 ( 0.14
0.32 ( 0.02
o-quinone
3,4-di-OHTOR-diSG
3,4-di-OHTOR-SG 2
3,4-di-OHTOR-SG 3
30
36
40
0.13 ( 0.05
0.03 ( 0.01
total: 0.48 ( 0.08
a
Incubations were conducted for 30 min with 0.1 mM substrate and rat liver microsomes (1.0 nmol of P450/mL) in the presence of an
NADPH-generating system and 2.5 mM [3H]GSH (specific activity of 40 nCi/nmol) at 37 °C. Radioactivity eluting from the HPLC column
was measured in fractions collected at 18 s intervals. Results are the average ( SD of three incubations.
Ta ble 3. Con ver sion of Dr oloxifen e to GSH Con ju ga tes by Ra t Liver Micr osom esa
retention
time (min)
rate of formation
quinoid
conjugate
[nmol (nmol P450)-1(10 min)-1
]
o-quinone
3,4-di-OHTAM-diSG 1
3,4-di-OHTAM-diSG 2
3,4-di-OHTAM-SG 1
3,4-di-OHTAM-SG 2
3,4-di-OHTAM-SG 3
29
31
34
37
41
0.025 ( 0.003
0.160 ( 0.016
0.057 ( 0.006
0.084 ( 0.004
0.084 ( 0.016
total: 0.41 ( 0.05
a
Incubations were conducted for 30 min with 0.1 mM substrate and rat liver microsomes (1.0 nmol of P450/mL) in the presence of an
NADPH-generating system and 2.5 mM [3H]GSH (specific activity of 40 nCi/nmol) at 37 °C. Radioactivity eluting from the HPLC column
was measured in fractions collected at 18 s intervals. Results are the average ( SD of three incubations.
stitution on the relative hepatotoxic effects of 4-methylphe-
nols showed that the toxic mechanism likely involves
biotransformation to a reactive quinone methide (47). The
data showed that the presence of electron-withdrawing
substituents (2-chloro or 2-bromo) markedly enhanced
both metabolism and toxicity of 4-methylphenol. As far
as o-quinone formation was concerned, droloxifene (Table
3) gave similar amounts of o-quinone-derived GSH

1650 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Yao et al.
the protonated molecule of 3,4-dihydroxytoremifene thy-
midine adduct. The ion at m/z 562 (100%) [MH - 116]+
represented the fragment resulting from loss of the
deoxyribosyl moiety from the protonated molecule. The
ion at m/z 72 (21%) was identified as dimethylaziridinium
[CH2CH2N(CH3)2+]. The tandem mass spectrum of the
3,4-dihydroxytoremifene deoxyguanosine adduct is shown
in Figure 5. The ion at m/z 703 (15%) was the protonated
molecule of the deoxyguanosine adduct. The ion at m/z
587 (100%) [MH - 116]+ represented the fragment
resulting from loss of the deoxyribosyl moiety from the
protonated molecule. The deoxyadenosine and deoxycy-
tosine adducts of toremifene-o-quinone were not isolated
and characterized because the yields were too low to be
detected by HPLC with UV detection. As a result, only
LC-MS-MS was used to analyze these adducts.
Three adducts were generated from reaction of thymi-
dine with toremifene-o-quinone (Figure 3). The major
adduct 3,4-di-OHTOR-T 1 (retention time 24.0 min) was
isolated and characterized. On the basis of 1H NMR
(Figure 6), COSY-NMR, and DEPT-NMR, we predict that
toremifene-o-quinone forms a covalent bond between the
C6 position on the o-quinone and N3 position of thymi-
dine (Figure 6). In the 1H NMR of 3,4-di-OHTOR-T 1,
the peak representing the proton attached to the N3
position of thymidine (δ ) 11.26 ppm) disappeared, which
suggested that the binding between thymidine and the
o-quinone occurred at this position. Thymidine was also
found to form adducts with 3,4-estrone-o-quinone at the
N3 position and the C1 position of the steroid (48). The
proton NMR spectrum also showed characteristic thy-
midine peaks for the C5 methyl group (1.91 ppm) and
the C6 proton (7.58 ppm). The significant difference
between the coupling pattern of aromatic protons of the
catechol ring in 3,4-dihydroxytormifene and 3,4-di-
OHOR-T 1 indicated that the reaction between toremifene-
o-quinone and thymidine occurred in the catechol ring
of the o-quinone. Proton NMR showed the typical meta-
coupling between the C2 and C6 protons of the 3,4-
dihydroxytoremifene moiety (J ≈ 2 Hz), which was also
observed in the COSY-NMR. This suggested that the
reaction occurred at the C5 position. DEPT-NMR showed
12 aromatic carbons attached to one proton, one from
thymidine (C6 position) and 11 from the 3,4-dihydroxy-
toremifene moiety which supports addition in the cat-
echol ring. We concluded that the 3,4-di-OHTOR-T 1 was
a Z-isomer based on its similarity to the proton NMR of
the Z-isomer of 3,4-dihydroxytoremifene glutathione
conjugates. In the proton NMR of the GSH conjugates,
the two meta-coupling aromatic protons in the catechol
ring of the Z-isomer (3,4-di-OHTOR-SG 1) had lower
chemical shifts (6.34 and 6.41 ppm) than those of the
E-isomer (3,4-di-OHTOR-SG 2) (6.75 and 6.81 ppm). The
four aromatic protons in the phenyl ring with the
dimethylaminoethoxy substituent of the Z-isomer had
higher chemical shifts (7.06 and 7.28 ppm) than those of
the E-isomer (6.68 and 6.86 ppm) (33, 34).
F igu r e 3. LC-MS-MS multiple reaction monitoring chromato-
grams of toremifene-o-quinone adducts with thymidine showing
the chlorine isotope pattern for each species including (a) 3,4-
di-OHTOR-T 1, (b) 3,4-di-OHTOR-T 2, (c) 3,4-di-OHTOR-T 3.
conjugates when compared to 4-hydroxytamoxifen and
4-hydroxytoremifene.
Rea ction of Tor em ifen e-o-qu in on e w ith Deoxy-
n u cleosid es. Toremifene-o-quinone was incubated with
four deoxynucleosides under physiological conditions, and
the solutions were analyzed by HPLC and LC-MS-MS.
Toremifene-o-quinone reacted with all four deoxynucleo-
sides to form the corresponding adducts. Three adducts
were detected in incubations with thymidine, five adducts
in incubations with deoxyguanosine, two adducts in
incubations with deoxyadenosine, and eight adducts in
incubations with deoxycytosine. Toremifene-o-quinone
produced considerably more adducts with thymidine as
compared to the other three deoxynucleosides. Our
previous work showed that tamoxifen-o-quinone formed
adducts with deoxyguanosine and thymidine, but not
with deoxyadenosine or deoxycytosine (34). As supported
by the kinetic data which showed that the toremifene-
o-quinone was considerably more reactive as compared
to the tamoxifen-o-quinone, we also observed that the
toremifene-o-quinone was much more effective at forming
deoxynucleoside adducts.
The toremifene-o-quinone deoxynucleoside adducts
were analyzed by LC-MS-MS. Three experiments were
used to provide evidence for these adducts. First, the
protonated molecule of each deoxynucleoside adduct was
detected by mass spectrometry. Second, MS-MS with CID
showed the product ion of [MH - 116]+ resulting from
loss of the sugar and the ion at m/z 72 corresponding to
dimethylaziridinium, CH2CH2N(CH3)2+. Finally, the iso-
topic ratios of the protonated molecule of each deoxy-
nucleoside adduct correlated with the natural abundance
ratio between 35Cl and 37Cl (3:1) (Figure 3). The MS-MS
spectrum of the 3,4-dihydroxytoremifene thymidine ad-
duct is shown in Figure 4. The ion at m/z 678 (22%) was
DNA adducts of tamoxifen and toremifene have been
detected in experimental animals (15, 49). Binding of
tamoxifen and toremifene to DNA in rat liver was studied
using 32P-postlabeling methods with HPLC-radioactivity
detection (24). Two major adducts and at least six minor
adducts were produced in the livers of female Sprague-
Dawley rats. Toremifene was found to produce only one
detectable adduct. The major tamoxifen adducts were
suggested to be R-(N2-deoxyguanosinyl)tamoxifen and

Droloxifene- and Toremifene-o-quinones
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1651
F igu r e 4. Positive ion electrospray MS-MS with CID of the protonated molecule of 3,4-di-OHTOR-T 1 at m/z 678.
F igu r e 5. Positive ion electrospray MS-MS with CID of the protonated molecule of 3,4-di-OHTOR-dG 1 at m/z 703.
R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen. The R-hy-
droxylation of the ethyl group is proposed to be the major
metabolic activation pathway of tamoxifen to form DNA
adducts (50). These tamoxifen-DNA adducts were also
identified in the endometrium of women treated with
tamoxifen (51). Since we have shown that both tamoxifen-
o-quinone and toremifene-o-quinone reacted with deoxy-
nucleosides to form corresponding adducts, the o-quinone
pathway might contribute to the genotoxicity of tamox-
ifen and toremifene.
Cytotoxicity Stu d ies in MDA-MB-231 a n d S30
Cells. We examined the relative toxicity of tamoxifen,
analogues, and metabolites in two breast cancer cell lines,
S30 (ER+) and MDA-MB-231 (ER-). The MDA-MB-231
cell line is an estrogen receptor negative cell line and the
S30 cell line is an estrogen receptor positive cell line
obtained from the MDA-MD-231 cell line stably trans-
fected with ERR. We have previously shown that ERR
positive cell lines are considerably more sensitive to the
toxic effects of equine catechol estrogens (39), and as a
result, it was of interest to determine if similar effects
were observed with the antiestrogens and metabolites.
Tamoxifen, 4-hydroxytamoxifen, droloxifene, 3,4-dihy-
droxytamoxifen, 4-hydroxytoremifene, and 3,4-dihydroxy-
toremifene were all shown to be cytotoxic in both cell lines
(Table 4). Droloxifene, 4-hydroxytamoxifen, and 4-hy-
droxytoremifene had equivalent cytotoxic potency in the
estrogen receptor negative cell line (MDA-MB-231) and
were more toxic than tamoxifen, 3,4-dihydroxytamoxifen,
and 3,4-dihydroxytoremifene in the MDA-MB-231 cell
line. Tamoxifen and 4-hydroxytamoxifen were more toxic
than droloxifene, 4-hydroxytoremifene, 3,4-dihydroxyta-
moxfen, and 3,4-dihydroxytoremifene in the estrogen
receptor positive cell line (S30). 3,4-Dihydroxytamoxifen
and 3,4-dihydroxytoremifene had equivalent cytotoxic
effects in both cell lines. Since the catechols showed less
cytotoxic effects in breast cancer cells compared to the
phenols, the catechol pathway may play a minor role in
cytotoxicity. However, the catechols might contribute to
the genotoxic effects of tamoxifen and toremifene since

1652 Chem. Res. Toxicol., Vol. 14, No. 12, 2001
Yao et al.
F igu r e 6. 1H NMR spectrum of 3,4-di-OHTOR-T 1.
Ta ble 4. Cytotoxicity of An tiestr ogen Meta bolites in
Br ea st Ca n cer Cell Lin esa
toremifene and droloxifene are considerably less effective
at induction of DNA adducts or liver tumors in animal
models compared to tamoxifen (7, 15, 23). As a result,
o-quinone formation from these antiestrogens likely
represents a minor genotoxic pathway in vivo.
LC50 (µM)
substrate
MDA-MB-231
S30
tamoxifen
4-OHTAM
droloxifene
4-OHTOR
3,4-di-OHTAM
3,4-di-OHTOR
34 ( 3
29 ( 3
23 ( 2
28 ( 3
37 ( 1
35 ( 3
23 ( 1
19 ( 2
32 ( 1
33 ( 2
40 ( 2
38 ( 3
Ack n ow led gm en t. This work is supported by NIH
Grants CA79870 and CA83124. We thank Dr. Yousheng
Hua for assistance with mass spectrometry experiments.
We also thank Dr. Shaonong Chen and Mr. J ianqiao Gu
for their help in the structure characterization of 3,4-
dihydroxytoremifene thymidine adduct. We acknowledge
Dr. J ohn M. Pezzuto for access to the Bioassay Research
Facility. We are grateful to Dr. V. C. J ordan (Northwest-
ern University) for the gift of the S30 cell line.
Cells (105 cells/mL) were incubated with various concentra-
tions of tested substrates for 18 h. Cell viability was determined
by trypan blue exclusion as described in Materials and Methods.
Results represent the mean ( SD of at least three determinations.
a
we have shown that tamoxifen-o-quinone (34) and
toremifene-o-quinone forms adducts with deoxynucleo-
sides.
Refer en ces
In conclusion, we have found that 3,4-dihydroxy-
toremifene was oxidized by silver oxide or tyrosinase to
an o-quinone which reacted with GSH giving the corre-
sponding conjugates. In microsomal incubations, both
4-hydroxytamoxifen and 4-hydroxytoremifene were me-
tabolized to quinone methides and o-quinones. In con-
trast, droloxifene was only transformed to an o-quinone
although the amount of o-quinone-derived GSH conju-
gates were similar as compared to 4-hydroxytamoxifen
and 4-hydroxytoremifene. Both the tamoxifen-o-quinone
and toremifene-o-quinone reacted with deoxynucleosides
to give corresponding adducts. However, the toremifene-
o-quinone was shown to be considerably more reactive
than the tamoxifen-o-quinone in terms of both kinetic
data as well as the yield and type of deoxynucleoside
adducts formed. In cytotoxicity studies in human breast
cancer cell lines, both 3,4-dihydroxytamoxifen and 3,4-
dihydroxytoremifene were less toxic than their parent
phenols, 4-hydroxytamoxifen and 4-hydroxytoremifene,
suggesting that o-quinone formation represents a minor
cytotoxic pathway. However, the fact that the o-quinones
formed adducts with deoxynucleosides in vitro implies
that the o-quinone pathway could contribute to the
genotoxicity of the antiestrogens in vivo. It should be
noted that the major genotoxic pathway for tamoxifen
likely involves R-hydroxylation, sulfate ester conjugation,
and loss of the sulfate group producing a carbocation
which forms covalent DNA adducts at the exocyclic amino
groups of adenine and guanine (52, 53). In addition, both
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